The overarching goal of this project is to develop a highly effective vaccine for prevention of Plasmodium falciparum (Pf) malaria based on a genetically attenuated (GA) triple gene knockout sporozoite (SPZ)-stage parasite that is aseptic, purified and safe for parenteral injection. In this Phase I application, we propose to optimize the manufacturing processes for the vaccine (Sanaria PfSPZ-GA2) and produce 1 lot suitable for clinical testing following Good Manufacturing Practices (GMPs). There is a high likelihood of success based on (1) the sterilizing immunity induced in humans immunized with attenuated PfSPZ (1st shown in 1970s) and (2) the tremendous progress achieved in manufacture and clinical testing of a 1st generation PfSPZ-based vaccine, called Sanaria PfSPZ Vaccine. In clinical trials in the U.S, Germany, Mali and Tanzania, PfSPZ Vaccine, which is attenuated by radiation rather than by discrete gene knockout, has induced >90% sterile protective immunity against controlled human infection (CHMI) conducted 3 weeks after immunization (5 clinical trials), and protection against heterologous CHMI at 8 months. Moreover, we documented sterile protection durable for at least 6 months against intense naturally transmitted Pf malaria in the field in Africa (2 clinical trials). PfSPZ Vaccine is being assessed in 7 clinical trials in the U.S., Germany and Africa, with licensure by the U.S. FDA anticipated in ~2.5 to 3 years. Importantly, PfSPZ Vaccine has proven exceptionally safe and well tolerated in all recipients (>1,100), including infants, children and adults. However, manufacture of PfSPZ Vaccine entails operator risks (PfSPZ are fully infectious prior to irradiation) and the genetic damage is not precisely characterized. For these reasons, we believe that there would be significant advantages, leading to major savings in cost of goods (COGs), if radiation-attenuated parasites were replaced with GA parasites. GA parasites are safer for operators (cannot cause malaria), the genetic defect is precisely characterized, and there may be improved in potency. Our colleagues at the Center for Infectious Disease Research demonstrated that eliminating the sap1, p52 and p36 genes leads to complete attenuation at the liver stage, similar to after radiation. This murine malaria triple knock-out elicited excellent protective immunity against SPZ challenge in rodents, and the Pf?p52?p36?sap1 SPZ construct, PfGAP3KO, was safe, well tolerated and immunogenic when administered to humans via mosquito bite, and did not cause breakthrough blood stage infections. Based on these encouraging data, we propose expedited development of aseptic, purified, vialed, cryopreserved ?p52?p36?sap1 PfSPZ. The vaccine (PfSPZ-GA2) will l be manufactured and quality control released in compliance with GMPs. Specific aims are to (1) optimize production parameters; (2) generate Master and Working Cell Banks; (3) manufacture 1 clinical lot. This will launch clinical development of the vaccine with the objective of licensing PfSPZ-GA2 for preventing malaria in travelers, for preventing pregnancy malaria and for mass vaccination programs (MVPs) aimed at regional malaria elimination.